P1648: THE RELATION BETWEEN THE SECONDARY STRUCTUE OF HEMOGLOBIN AND ENHANCED HYPOXIA-INDUCED ATP RELEASE FROM RED BLOOD CELLS IN APOE/LDLR DOUBLE-DEFICIENT MICE
Authors: Fatih Celal Alcicek, Jakub Dybas, Aneta Blat, Katarzyna Bulat, Tasnim Mohaissen, Deniz Yildirim Dogan, Isabelle Hornung, Wiktoria Rutkowska, Andrea U. Steinbicker, Katarzyna M. Marzec
Journal: HemaSphere (2023)

Topic: 34. Thrombosis and vascular biology - Biology & Translational Research Background: Red blood cells (RBCs) play a role in the regulation of vascular tone via release of adenosine triphosphate (ATP) into the vasculature. ATP release occurs in response to various stimuli, e.g., lower oxygen tension, and leads vasodilation and increased blood flow. Interestingly, ApoE/LDLR double deficient (ApoE/LDLR-/-) mice, a mouse model of atherosclerosis, display higher exercise capacity compared to age-matched controls. However, it is not known whether increased exercise capacity in ApoE/LDLR-/- mice is linked to induced ATP release from RBCs. Aims: The aim of this work is to evaluate how affected the induced ATP release from RBCs, the secondary structure of hemoglobin (Hb), and 2,3-diphosphoglycerate (2,3-DPG) levels in RBCs in ApoE/LDLR double deficient mice with the change of age and sex. Methods: Murine model of atherosclerosis, ApoE/LDLR-/-, and wild-type controls, C57BL/6J, were used for the experiments (8 and 24 weeks old, female and male, N = 4-13). Whole blood samples were collected, and RBCs were isolated for the experiments. RBCs were washed and suspended with the daily buffer solution (DBS). The DBS was prepared on the same day for each experiment, and it contained: 21 mM Tris Base, 140.5 mM NaCl, 4.7 mM KCl, 2 mM CaCl2, 1.2 mM MgSO4, 5.5 mM glucose and 76 µM bovine serum albumin, with a final pH adjusted to 7.40. All chemicals were dissolved in distilled water and the solution was filtered through a pleated filter with 0.22 µm pore size. To induce ATP release from RBCs, 3V cocktail (10 µM isoproterenol, 30 µM forskolin, and 100 µM papaverine) and hypoxia were applied at 37°C for 10 and 5 minutes, respectively. For inhibition of PANX1 channel, isolated RBCs were incubated with 10 µM carbenoxolone (CBX) at 37°C for 5 minutes prior to the application of 3V and hypoxia, respectively. Attenuated total reflectance - Fourier transform infrared (ATR-FTIR) spectroscopy performed to investigate the secondary structure alterations in Hb. Intracellular 2,3-DPG levels were measured using a commercially available ELISA kit on RBCs subjected to subsequent hemolysis according to the manufacturer’s instructions. Results: The results indicate notable changes in the secondary structure of Hb and higher 2,3-DPG levels in RBCs from ApoE/LDLR-/- mice compared to their age-matched controls. This may suggest that alterations in Hb structure in ApoE/LDLR-/- mice are irreversible. 3V-induced ATP release from RBCs occurred at comparable levels for all groups, which indicates the activity of adenylyl cyclase and the components of upstream signal-transduction pathway are intact. Moreover, hypoxia-induced ATP release from RBCs was higher in ApoE/LDLR-/- mice compared to their age-matched controls, a potential contributing factor and a finding in line with the higher exercise capacity. Summary/Conclusion: Taken together, augmented hypoxia-induced ATP release from RBCs in ApoE/LDLR-/- mice indicates a possible altered component in the ATP release pathway. Previously, the role of confirmational change of Hb in hypoxia-induced ATP release from RBCs had been reported. We suggest that the altered Hb structure, or another component related, could lead to the exhibited results in ApoE/LDLR-/- mice. Further studies to characterize the altered components of the ATP release mechanism in ApoE/LDLR-/- could lead to pharmacological modification of pathologically impaired microcirculation. Keywords: Atherosclerosis, Red blood cell, Hemoglobin